The present invention relates to lactobacilli able to inhibit the growth of pathogenic microorganisms and/or to exert a microbicidal activity against them (more particularly two novel Lactobacillus gasseri strains and one Lactobacillus crispatus strain) and a method for inducing and/or enhancing and/or keeping said activity in lactobacilli cultures.
The invention also relates to a culture medium for lactobacilli which can be used in combination with said induction method.
Lactobacilli are widespread in nature. A number of species are present in alimentary products (milk, yogurt, fruits, vegetables and the like), whereas only some strains are usual eubiotic saprophytes of the intestinal and/or urogenital flora.
In most cases known lactobacilli, when administered through the oral or vaginal or any other routes, restore the physiological bacterial flora, but are per se not able of inhibiting the growth of pathogens or of exerting any microbicidal activity against them.
Only some bacterial strains have recognizedly developed this natural capability of inhibiting the growth of pathogenic microorganisms when placed in the same culture.
In fact, EP 0 353 581 reports that a Lactobacillus fermentum strain, deposited at the CNCM of the Institut Pasteur, recovered from human vagina, is capable of counteracting in vitro the growth of Candida albicans. 
Furthermore some other lactobacilli, in particular a Lactobacillus paracasei strain recovered from human intestine (Italian Pat. Appl. No MI97A 000426 filed on Feb. 27, 1997), have been found to have, in addition to the other features cited in the above mentioned Patent Application, the capability of causing in vitro an inhibition area when seeded in agarized Petri dishes in which Candida albicans had concomitantly been grown.
As a whole, these findings prove that the activity of lactobacilli, recovered from various human habitats, against pathogens, occurs naturally.
Moreover, such an antimicrobial activity is known to be related to the ability of some lactobacilli to physiologically produce bacteriocins and/or other similar substances, said production of bacteriocins being sometimes related to the presence of plasmids.
The stabilization of an industrially important phenotypic character in lactobacilli by addition of specific substances has been described in GB 1 560 208, wherein the capability of producing lactic acid was induced in lactobacilli.
It has now been found that only some lactobacilli, recovered from human physiological habitats, when seeded in Petri dishes containing agarized medium in which some pathogens had previously been seeded and grown, exert an inhibitory or microbicidal action against such pathogens.
These properties are related to the ability of some lactobacilli to physiologically produce bacteriocins and/or, other similar substances, having bactericidal or microbicidal action. However, such bacterial strains having these characteristics can effectively be used in therapy only if they keep and/or increase said properties in time. It is moreover desirable to induce the inhibitory activity against pathogens in lactobacilli strains which are not per se capable of exerting said activity.
The ability of lactobacilli to produce bacteriocins and/or, other similar substances, is affected by a number of technical factors such as the composition of the culture medium and the incubation conditions (Temperature, pH, Oxygen).
Moreover, any antimicrobial substances produced are generally detected in laboratory conditions which are quite different from the original ones and in which the specific genetic determinants are usually constitutively expressed. Inducible genes are hardly detected by the usual laboratory assays.
The present invention relates to a method for producing lactobacilli strains having microbicidal activity useful for the therapeutic and/or prophylactic treatment of pathologies of the gastrointestinal and/or urogenital systems in humans.
The method further provides for maintaining or increasing the ability to produce antimicrobial substances in those lactobacilli strains which already have these properties, and even to induce and detect the said ability in those lactobacilli which, although having such an ability, cannot exert it.
Preferably, the lactobacilli strains subject to this treatment derive from the physiological intestinal and/or vaginal bacterial flora of a healthy human, and are typically obtained from biological samples containing a multiplicity of bacterial species, isolated by conventional techniques.
Before being treated according to the process of the invention, said samples are usually subjected to suitable pre-treatments according to conventional methods in order to maintain the strains until use.
It is an object of the present invention to provide a method for stimulating the cultures, traditionally prepared as described above, to produce specific antimicrobial substances.
The lactobacilli strains to be treated are contacted with the pathogenic strain against which the antimicrobial action has to be induced, or with its supernatant (where supernatant means the liquid, sterilised and stored in refrigerator or in freezer until use, which separates from the cell pellet upon centrifugation of a pathogenic culture which is obtained growing a strain of the appropriate pathogen in a suitable culture medium and under suitable growth conditions).
Alternatively, the culture medium for lactobacilli can also be contacted with cells of pathogenic microorganisms inactivated by chemical or physical treatments.
After that the, lactobacilli and pathogenic strain or its supernatant are seeded and grown simultaneously in the suitable culture medium. This step of incubation of the strains is typically carried out on a plate, in a nutritive medium added with agar, preferably on MRS agarized medium.
The resulting lactobacilli strains are then centrifuged and resuspended in a medium containing the specific excipients, then frozen so as to keep the strain viable.
The present invention further relates to lactobacilli strains and the bacterial cultures obtained according to the process of this invention, and to compositions comprising pharmaceutical, dietary or alimentary formulations containing the lactobacilli as the principal active ingredient together with a pharmaceutically or physiologically acceptable carrier.
The process of the invention provides lactobacilli strains able to inhibit different pathogens and/or exert a microbicidal action against them, suitable for formulation in pharmaceutical preparations, which proved to be extremely effective and advantageous in the treatment of pathologies related to the presence of pathogenic agents sensitive to some lactobacilli strains.
According to a preferred embodiment of the present invention, the bacterial strains to be treated are derived from the physiological saprophytic flora of healthy subjects.
In order to activate the inducible genes of lactobacilli to produce antimicrobial substances, the bacterial cultures are contacted with cultures of pathogenic strains such as Candida sp., Proteus sp., Escherichia coli, Trichomonas vaginalis, Streptcoccus xcex2-haemolyticus, Enterococcus sp, or with the supernatants thereof.
xe2x80x9cSupernatantxe2x80x9d means the culture medium (for example LB broth for Escherichia coli, or Malt Broth for Candida sp.) in which a culture of the pathogenic strain has been grown in the suitable growth conditions (for example 18 hours in aerobic conditions at 37xc2x0 C. for Escherichia coli, and 24 hours in aerobic conditions at 30xc2x0 C. for Candida). At the end of the growth period described above, the culture is centrifuged and the liquid separated from the cell pellet is withdrawn. This liquid is then sterilised (for example by filtration through cellulose filters with porosity not higher than 0.45 micron) and stored in refrigerator or freezer until use.
Alternatively to the addition of cultures of pathogens or of supernatants thereof, the lactobacilli culture medium can also be added with cells of pathogenic microorganisms inactivated by chemical (chloroform, formaldehyde) or physical (heat or radiations) treatments.
According to a preferred embodiment of the invention, the lactobacilli strain to be treated is grown, at the same time as the pathogenic strain, in a lactobacilli medium, preferably MRS medium (de Man J. C., Rogosa, M. and M. E. Sharpe, Journal of Applied Bacteriology, 23, 130-135, 1960) the composition of which is reported in the subsequent table.
Subsequently, the strains inoculated in the test-tubes tubes containing the MRS medium described above are incubated in thermo-stat at +37xc2x0 C., in anaerobiosis conditions.
The lactobacilli strains are grown in these optimum growth conditions for 24 hours in liquid MRS, then they are centrifuged and resuspended, preferably in 0.5 ml of liquid MRS, containing a varying concentration of glycerol, preferably ranging from 20 to 50%. This procedure keeps the treated strain viable.
Laboratory assays prove that the addition of either supernatant liquid, as defined above, in a 0.01% to 5.00% V/V ratio, or of the pathogenic strain to the lactobacilli culture medium, increases the production of inhibiting substances.
The following examples further illustrate the invention.